miRNA-101抑制A549细胞增殖作用机制探讨

目的 探讨miR-101在非小细胞肺癌(NSCLC)增殖中的作用及机制.方法 通过实时荧光定量聚合酶链反应(qRT-PCR)检测20例NSCLC组织、癌旁组织及NSCLC细胞系A549、H1650、H1975、PC-9和BEAS-2B人正常肺上皮细胞中miR-101的表达.使用miR-101的类似物miR-101-mi...

Effect of miRNA-101 on inhibition the proliferation of A549 cells by targeted regulation of SOX2 expression

Objective To detect the expression and role of miR-101 in non-small cell lung cancer(NSCLC)and explore its possible mechanism.Methods The expression of miR-101 in NSCLC tissues,paracancerous tissues and NSCLC cell lines A549,H1650,H1975,PC-9 and normal lung epithelial cells BEAS-2 Bwere detected by quantitative real-time polymerase chain reaction(qRT-PCR).A549 cells were transiently transfected with miR-101 analogues(miR-101-mimics)and the control(miR-NC),and detected the cell proliferation by MTT,cell cycle and apoptosis by flow cytometry and the expression of apoptosis-related proteins Bcl-2,Bax,Cleaved Caspase-3,cleaved-caspase9 by western blotting and then detected the Luciferase activity to confirmed miR-101 targeted regulation the expression of SOX2.The expression of SOX2 in NSCLC tissues and adjacent tissues were detected by immunohistochemistry and qRT-PCR.The correlation between the expression of SOX2 and miR-101 was analyzed by Pearson correlation coefficient in NSCLC tissues.Results The expression of miR-101 in NSCLC tissues(0.29±0.11)was lower than adjacent tissues(0.87±0.08),the mean difference between groups was statistically significant(t=12.54,P=0.0001).The absorbance(A)values of A549 cells transfected with miR-101-mimic and miR-NC were 0.43±0.04 and 0.65±0.05 at 24 hours respectively,the mean difference between groups was statistically significant(t=3.033,P=0.0387).The A values of A549 cells transfected with MiR-101-mimic and miR-NC were 0.52±0.05 and 0.76±0.05 at 48 hours respectively,the mean difference between groups was statistically significant(t=7.368,P=0.0018).The present of s phase cells was increased in miR-101-mimic transfected A549 group,and the value of miR-101-mimic group and miR-NC group were(23.43±0.90)%and(9.80±1.32)%respectively,the mean difference of between groups was statistically significant(t=14.75,P=0.0001).The apoptosis rate of A549 cells were(22.19±1.92)%and(7.58±1.21)%in miR-101-mimic group and miR-NC group respectively,which was statistically significant(t=14.23,P<0.0001).The expression of apoptotic-related protein cleaved caspase-9,cleaved caspase-9 and bax was increased,while the expression of anti-apoptotic protein Bcl-2 was decreased.The relative luciferase activities of miR-NC+WT,miR-101+WT,miR-NC+MU1,miR-101+MU1,miRNA-NC+MU2,miR-101+MU2 were 1.003±0.025,0.253±0.015,1.017±0.025,0.510±0.006,0.997±0.021 and 0.417±0.067.Compared with miR-NC+WT,the luciferase activity of miR-101+WT group was decreased,and the mean difference between groups was statistically significant(t=44.13,P<0.0001).Compared with miRNA-NC+MU1,the luciferase activity in miRNA-101+MU1 group was decreased,and the mean difference between groups was statistically significant(t=13.33,P=0.0002).Compared with miR-NC+MU2,the luciferase activity of miR-101+MU2 group was decreased,and the mean difference between groups was statistically significant(t=14.40,P=0.0001).Compared with miR-101+WT,the luciferase activity of miR-101+MU1 group was increased,and the difference of mean value between groups was statistically significant(t=7.088,P=0.0021).Compared with miR-101+WT,the activity of luciferase in miRNA-101+MU2 group was increased,and the difference of mean value between groups was statistically significant(t=4.141,P=0.0144).The relative expression values of SOX2 in miR-101-mimic Group and miR-NC group were 1.331±0.069 and 0.537±0.034 respectively,the mean difference between the two groups was statistically significant(t=17.72,P=0.0001).The expression of SOX2 protein was detected by western blotting,and the overexpression of miR-101 decreased the expression of SOX2 protein.The positive rate of SOX2 was 80.0%(16/20)and 25.0%(5/20),in lung cancer tissues and adjacent tissues respectively.The difference was statistically significant(χ²=12.13,P=0.0005).The relative expression of SOX2 mRNA in lung cancer tissues and adjacent tissues were 0.267±0.018 and 0.894±0.347,the mean difference between the two groups was statistically significant(t=5.705,P<0.0001).The expression of SOX2 was negatively correlated with miR-101,R²=0.9354.Conclusions miR-101 can effectively inhibit the proliferation and induce apoptosis of lung cancer A549 cells.Its possible mechanism is targeted inhibition of SOX2 expression.